title: Temperature dependence of binding and catalysis for human serum arylesterase/paraoxonase.
abstract: The influence of temperature upon the hydrolysis of phenyl acetate, catalysed by purified human serum arylesterase/paraoxonase (E. C. 3.1.8.1), was studied in the temperature range 10 °C-40 °C by spectrophotometry in TRIS buffer, pH 8.0, using both initial rate analysis and progress curve analysis. The kinetic parameters (catalytic constant k(cat); Michaelis constant K(m); product inhibition constant K(p)) were determined by nonlinear regression. All parameters increased with temperature, but the ratios k(cat)/K(m) and K(p)/K(m) remained practically constant. Binding of both substrate and reaction product (phenol) was exothermic. A negative entropic term accounted for about 50% of the enthalpy change for both the binding and catalytic steps. Thermodynamic analysis suggested that: (1) the rate-limiting step is the nucleophilic attack of the carbonyl group of the substrate by a water molecule, (2) the active site is preorganized with no induced fit, (3) the enzyme-bound calcium plays an important role in stabilizing both the substrate and the transition state. The practical implications of these results are discussed.
authors.affiliation: Service de Pharmacologie-Toxicologie, Hôpital Dupuytren, 2 Avenue Martin Luther King, 87042 Limoges, France. Electronic address: jean.debord@unilim.fr.
authors.forename: Jean
authors.initials: J
authors.lastname: Debord
authors.affiliation: Groupement de Recherche Eau, Sol, Environnement, Université de Limoges, Faculté des Sciences, 123 Avenue Albert Thomas, 87060 Limoges, France.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Bollinger
authors.affiliation: Institut de Mathématiques de Toulouse (UMR CNRS 5219), 31062 Toulouse, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Harel
authors.affiliation: Service de Gérontologie clinique, Hôpital Dupuytren, 2 Avenue Martin Luther King, 87042 Limoges, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Dantoine
mesh_terms: D000085:Acetates / Q000737:chemistry*; D043303:Aryldialkylphosphatase / Q000737:chemistry* / Q000302:isolation & purification; D001665:Binding Sites; D055162:Biocatalysis; D001798:Blood Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D010636:Phenols / Q000737:chemistry*; D012996:Solutions; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D014867:Water
chemical_list: D000085:Acetates; D001798:Blood Proteins; D007527:Isoenzymes; D010636:Phenols; D012996:Solutions; D014867:Water; C570634:phenyl acetate; D043303:Aryldialkylphosphatase
keywords: Activation parameters; Arylesterase; Integrated Michaelis equation; Paraoxonase; Product inhibition